Distinct modes of action of IAPP oligomers on membranes

Islet Amyloid Polypeptide (IAPP, also known as amylin) is a peptide hormone which is co-secreted with insulin by pancreatic β-cells and forms amyloid aggregates in type II diabetes. Various lines of evidence indicate that oligomers of this peptide may induce toxicity by disrupting or forming pores in cell membranes but the structures of these pores are unknown. Here we create models of pores for both helical and β-structured peptides using implicit membrane modeling and test their stability using multimicrosecond all-atom simulations. We find that the helical peptides behave similarly to antimicrobial peptides; they remain stably inserted in a highly tilted or partially unfolded configuration creating a narrow water channel. Parallel helix orientation creates a somewhat larger pore. An octameric β barrel of parallel β-hairpins is highly stable in the membrane, whereas the corresponding barrel made of antiparallel hairpins is not. We propose that certain experiments probe the helical pore state while others probe the β-structured pore state; this provides a possible explanation for lack of correlation that is sometimes observed between in vivo toxicity and in vitro liposome permeabilization experiments.

The functional regulatory details of ERK2 in complex with RSK1: an in silico insight

DFG, αC-helix orientation regarding the active site position and distance between K54 and Glu71 in the active and inactive states of ERK2.

Comparing Interfacial Trp, Interfacial His and pH Dependence for the Anchoring of Tilted Transmembrane Helical Peptides

Charged and aromatic amino acid residues, being enriched toward the terminals of membrane-spanning helices in membrane proteins, help to stabilize particular transmembrane orientations. Among them, histidine is aromatic and can be positively charge at low pH. To enable investigations of the underlying protein-lipid interactions, we have examined the effects of single or pairs of interfacial histidine residues using the constructive low-dynamic GWALP23 (acetyl-GG2ALW5LALALALALALALW19LAG22A-amide) peptide framework by incorporating individual or paired histidines at locations 2, 5, 19 or 22. Analysis of helix orientation by means of solid-state 2H NMR spectra of labeled alanine residues reveals marked differences with H2,22 compared to W2,22. Nevertheless, the properties of membrane-spanning H2,22WALP23 helices show little pH dependence and are similar to those having Gly, Arg or Lys at positions 2 and 22. The presence of H5 or H19 influences the helix rotational preference but not the tilt magnitude. H5 affects the helical integrity, as residue 7 unwinds from the core helix; yet once again the helix orientation and dynamic properties show little sensitivity to pH. The overall results reveal that the detailed properties of transmembrane helices depend upon the precise locations of interfacial histidine residues.

High-resolution models of actin-bound myosin from EPR of a bifunctional spin label

We have employed two complementary high-resolution electron paramagnetic resonance (EPR) techniques with a bifunctional spin label (BSL) to test and refine protein structural models based on crystal structures and cryo-EM. We demonstrate this approach by investigating the effects of nucleotide binding on the structure of myosin’s catalytic domain (CD), while myosin is in complex with actin. Unlike conventional spin labels attached to single Cys, BSL reacts with a pair of Cys; in this study, we thoroughly characterize BSL’s rigid, highly stereoselective attachment to protein α-helices, which permits accurate measurements of orientation and distance. Distance constraints were obtained from double electron-electron resonance (DEER) on myosin constructs labeled with BSL specifically at two sites. Constraints for orientation of individual helices were obtained previously from continuous-wave EPR (CW-EPR) of myosin labeled at specific sites with BSL in oriented muscle fibers. We have shown previously that CW-EPR of BSL quantifies helix orientation within actin-bound myosin; here we show that the addition of high-resolution distance constraints by DEER alleviates remaining spatial ambiguity, allowing for direct testing and refinement of atomic structural models. This approach is applicable to any orientable complex (e.g., membranes or filaments) in which site-specific di- Cys mutation is feasible.

High-resolution helix orientation in actin-bound myosin determined with a bifunctional spin label

Using electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) bound stereospecifically to Dictyostelium myosin II, we determined with high resolution the orientation of individual structural elements in the catalytic domain while myosin is in complex with actin. BSL was attached to a pair of engineered cysteine side chains four residues apart on known α-helical segments, within a construct of the myosin catalytic domain that lacks other reactive cysteines. EPR spectra of BSL-myosin bound to actin in oriented muscle fibers showed sharp three-line spectra, indicating a well-defined orientation relative to the actin filament axis. Spectral analysis indicated that orientation of the spin label can be determined within <2.1° accuracy, and comparison with existing structural data in the absence of nucleotide indicates that helix orientation can also be determined with <4.2° accuracy. We used this approach to examine the crucial ADP release step in myosin’s catalytic cycle and detected reversible rotations of two helices in actin-bound myosin in response to ADP binding and dissociation. One of these rotations has not been observed in myosin-only crystal structures.

Activation helix orientation of the estrogen receptor is mediated by receptor dimerization: evidence from molecular dynamics simulations

ERα dimer formation reshapes the helix 12 conformational landscape and is a leading factor for the activation helix conformation.